49026 Milmont Drive, Fremont, CA 94538 Emergo Europe, Molenstraat 15, NL-2513 BH The Hague, The Netherlands

Tel : +1 (800) 421-4149, Fax: +1 (510) 824-1490,

support@biogenex.com

SPECIAL STAINS
GiemsaStaining
Cat. No. SS016
Doc. No. 932-SS016 Rev. No: B
Release Date: 06-Nov-2015

Intended Use
This product is intended for in vitro diagnostic use on the i6000  BioGenex Automated Staining System & also for Manual
staining.

Summary and Explanation

It is recognized that Helicobacter pylori is the cause of most stomach and duodenal ulcers. Approximately 95% of persons with
gastric ulcers and 100% of persons with chronic gastritis have this bacterium within the stomach. The organism has not been
found in healthy persons, those without stomach ulcers or gastritis. H. pylori is known to bind to the O blood-group structure (a
particular series of sugars) present on gastric epithelial cells.

Principles of the Procedure

Romanowsky dyes, or stains, contain a mixture of the basic dye, methylene blue, and the acid dye, eosin. These are
polychromatic stains that provide a wide color range when staining tissues and blood smears. Polychromatic stains were initially
named by their ability to form new substances-metachromatic dyes-particularly on standing in solution or at an alkaline pH.
Since standardization is currently important for histological procedures, Giemsa stains are now prepared using weighed amounts
of azures.

Reagents Provided

Giemsa Solution I (HX953-YAK) 1 x 20 mL, ready to use

Giemsa Solution II (HX954-YAK) 1 x 20 mL, ready to use
Giemsa Differentiation Solution (HX955-YAK) 1 x 20 mL, ready to use

Reagents Required but Not Supplied

EZ-DeWax™ Solution (HK584-5K) 1x500ml, concentrate
TM
Super Sensitive Wash Buffer, 20X (HK583-5K) 1x500ml, concentrate
Mounting Medium
Positive Control Tissue

Storage and Handling

Store all reagents at 2-8°C. Do not use after expiration dates as indicated on the reagent labels

Specimen Preparation

Fixation plays an important role in preserving the tissue structures to be visualized using this stain. 10% neutral buffered
formalin is the preferred fixative, however, other fixatives like Carnoy’s may also be used. Ensure that the fixed sections are
adequately embedded in paraffin. Cut tissue sections to 4-5 microns.

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 49026 Milmont Drive, Fremont, CA 94538 Emergo Europe, Molenstraat 15, NL-2513 BH The Hague, The Netherlands
Tel : +1 (800) 421-4149, Fax: +1 (510) 824-1490,
support@biogenex.com

Precautions
Not regarded as a health or environmental hazard under current legislation. This product contains no hazardous material at a
reportable concentration according to U.S.29 CFR 1910.1200 and EC Directive 91/155/EC.

BioGenex i6000 Automated Staining System Protocol

1. Bring reagents to room temperature. Deparaffinize the tissues and hydrate to distilled water.
2. Load the barcode-labeled slides for the appropriate stain into the BioGenex Automated Staining System slide racks. Load
the barcode-labeled reagent vials into the BioGenex Automated Staining System reagent racks.
3. Start Special Stains software on the instrument and check the protocol parameters against the factory default settings listed
below. Change any of the parameters as required. Please note that any parameter change, once saved, becomes the default
setting until changed again. Use of parameter settings other than factory default requires validation by the user.
4. Select “Start Scan” and the instrument will perform the steps listed in the table (if the factory default settings have been
selected).

Giemsa TrichromeStaining Protocol Default Setting:

Reagent No. of Incubations Incubation Time No. of After Rinses
Special Stains Wash Buffer 3 30 sec. 0
Giemsa I 1 3 min. 0
Giemsa II 1 3 min. 3
Giemsa Differentiation Soln 1 3 min. 3*
* blow steps after the wash steps

Mounting Instructions
After staining, dehydrate and clear in xylene as usual and apply permanent mounting medium.

Manual Protocol for Alcian Blue PAS Stain

1. Deparaffinize and hydrate the slides to distilled water.

2. Apply 250µl Giemsa solution I for 3 minutes. Blot the slide
3. Add 250µl Giemsa solution II and let stand for 3 minutes. Rinse in distilled water.
4. Add 250µl Giemsa differentiation solution and let stand for 3 minutes. Rinse in distilled water.
5. Dehydrate, clear, and coverslip

Quality Control
A positive control slide—one that will display positive staining with this stain—should be included in every run.

Trouble shooting
1. Follow data sheet instructions correctly.
2. Gently invert all reagents prior to use.
3. Use the correct parameters as given in the table above

Expected Results
Helicobacter pylori and nuclei stain dark blue, and cytoplasm stains pink

Limitations of the procedure

1. The thickness of the section may affect the intensity of the staining. Necrotic tissue may exhibit non-specific staining.
2. Use tissue, which shows positive structure or element for which it is being tested.
Performance Characteristics

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 49026 Milmont Drive, Fremont, CA 94538 Emergo Europe, Molenstraat 15, NL-2513 BH The Hague, The Netherlands
Tel : +1 (800) 421-4149, Fax: +1 (510) 824-1490,
support@biogenex.com

Specificity of Special Stain

The Giemsa staining procedure is specific for histological staining of Helicobacter pylori in tissue sections.
BioGenex has conducted studies to evaluate the performance of all its Special Stains products. BioGenex Special Stains have
shown reproducible and consistent results when used within a single run, between runs and between lots. The products have
been determined to be stable for the periods specified on the labels either by standard real time or accelerated testing methods.
BioGenex ensures product quality through 100% quality control for all products released and through surveillance programs.

Bibliography
1. Carson FL: Histotechnology: A self Instructional Text, ASCP Press, Chicago (IL), 1990:191